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Blood, Vol. 109, Issue 11, 4742-4752, June 1, 2007
Lack of endothelial cell survivin causes embryonic defects in angiogenesis, cardiogenesis, and neural tube closure
Blood Zwerts et al.
109: 4742
Supplemental materials for: Zwerts et al, Vol 109, Issue 11, 4742-4752
Files in this Data Supplement:
- Figure S1. Expression of survivin in vascular endothelium of embryos (PDF, 30.6 KB) -
Transverse histologic sections of E10.5 wild-type embryos were stained with antithrombomodulin (anti-TM) antibodies (A) and antisurvivin antibodies (B), and the confocal images were overlaid (C). In this representative view of the vasculature throughout the embryo, the endothelial cells of the dorsal aorta express survivin.
- Figure S2. Endothelial-specific functional expression of cre recombinase in embryos (PDF, 108 KB) -
Transverse sections of E10.5 embryos at the level of the midbrain were stained with antibodies directed against cre recombinase (A-B) or survivin (C-E). In tie1-cre/survivinlox/wt embryos (A-B), cre expression is restricted specifically to the vasculature (A) (cardinal vein shown, with arrows indicating positive staining), and to the endocardium (not shown), but is not detected in neural cells in the neuroepithelium (B) surrounding the neural tube at the midbrain. Boxes in top right corners of panels A and B represent high-power views of cre-expressing endothelium (A) and nonstained neuroepithelium (B). (C-E) Endothelial cell survivin expression is reduced or absent in cardinal vein endothelium of E10.5 tie1-cre/survivinlox/lox (D-E) embryos compared with endothelial expression in survivinlox/lox (C) embryos (arrows indicate prominent endothelial cell expression of survivin).
- Figure S3. Genotyping of tail DNA and embryos (PDF, 41.4 KB) -
(A) Diagrammatic representation of the wild-type survivin gene with 4 exons, the survivin floxed targeted allele, and the cre-excised floxed allele. Positions of oligonucleotide primers (Adv) used for genotyping are shown. (B) PCRs on tail DNA, with the different primer pairs shown in panel A, were used to genotype E10.5 embryos resulting from breeding tie1-cre/survivinlox/wt mice with survivinlox/lox mice. PCR using primer pair Adv17 (sense 5′-CAGGCCGATGGTCTCAGAAATA-3′) and Adv18 (5′-GGTTTCCTTCTTGCTATTCTGACT-3′) results in amplicons of 408 bp and 364 bp in the targeted allele across the 5′ loxP site and the wild-type allele, respectively. PCR with primer pair Adv25 (5′-GATGGTGATGAAACTAGCATCTCACCCTG-3′) and Adv28 (antisense 5′-GCTTAAGTCCACGTCACAATAGAGC-3′) results in a 577-bp amplicon across the 3′ loxP site, and a 386-bp amplicon in the wild-type allele. PCR using primers ADV17 and ADV28, which flank the floxed survivin gene, yields an amplicon of 425 bp when the survivin gene is cre excised. Presence of the transgene was confirmed by detection of a 447-bp amplicon generated by PCR using primer pair Cre403 (5′-GATGCCGGTGAACGTGCAAAACAGGCTC-3′) and Cre850 (antisense 5′-CGCCGTAAATCAATCGATGAGTTGCTTC-3′). Therefore, the genotypes of the embryos in lanes 1, 2, 3, and 4 (at bottom of panel B) are survivinlox/wt, tie1-cre:survivinlox/wt, survivinlox/lox, and tie1-cre:survivinlox/lox, respectively.
- Figure S4. Detection of hypoxia in embryos (PDF, 37.7 KB) -
E10.5 embryos were evaluated for evidence of hypoxia after intravenous pimonidazole injection into the pregnant mother and immunodetection of transverse sections through the mid- and hindbrain regions with the hydroxprobeTM-1 kit (“Detection of apoptosis and hypoxia”). Hypoxic regions (arrows) were detected in the neuroectoderm of tie1-cre:survivinlox/lox (–/–) embryos, but were not present in the corresponding regions of survivinlox/lox (+/+) embryos.
- Figure S5. Increased permeability of survivin+/– endothelial cells (PDF, 11 KB) -
Equal numbers of survivin+/+ (solid line) or survivin+/– (dashed line) endothelial cells (250 000 per well) were cultured in the top chamber of a 6-well transwell, and permeability to dextran-FITC (Mr 4300) was quantified spectrophotometrically at 480/530 nm at regular intervals.1 Permeability to survivin+/– cells is significantly increased at most time intervals (*P < .05). Results are representative of 2 separate experiments, and reflect the means of a study performed in triplicate.
- Figure S6. Scoring scheme for embryo neural tube closure in ex vivo cultures (PDF, 12.9 KB) -
Embryos were cultured for 24 hours ex vivo starting at E8.5 and were scored 0 to 5 according to the extent of neural tube closure, details of which are provided in “Coculture of embryos with conditioned media.” A side-view diagrammatic representation of the normal steps of neural tube closure is shown (adapted from Macdonald et al2 and reprinted with permission of Wiley-Liss, Inc, a subsidiary of John Wiley & Sons, Inc). Numbers 1 to 4 represent the order of contact sites or “closures,” and the arrows indicate the direction of fusion extending from each initial contact. P indicates prosencephalon; M mesencephalon; R, rhombencephalon; and C, cervical region.
REFERENCES
1. Lee TH, Avraham HK, Jiang S, Avraham S. Vascular endothelial growth factor modulates the transendothelial migration of MDA-MB-231 breast cancer cells through regulation of brain microvascular endothelial cell permeability. J Biol Chem. 2003;278:5277-5284.
2. Macdonald KB, Juriloff DM, Harris MJ. Developmental study of neural tube closure in a mouse stock with a high incidence of exencephaly. Teratology. 1989;39:195-213.
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